高山离子芥冷诱导基因转化甘蔗二元植物表达载体构建

Construction of bivalent plant expression vector with cold-induced gene of Chorispora bungeana in sugarcane

【目的】利用载体重组技术分别将高山离子芥冷诱导基因(Cbcor15a)和报告基因eGFP插入载体pCambia1300-bar,并引入玉米泛素基因启动子UBi-1代替载体本身启动子CaMV35s,重组为适合甘蔗转基因的二元植物表达载体pCambia1300-cbcor15a-bar。【方法】参照pCambia1300-bar载体多克隆位点和基因Cbcor15a、eGFP和启动子UBi-1的核苷酸序列设计引物,通过载体重组技术将基因和启动子分别插入相应的位点。利用基因枪分别将pCambia1300-bar载体和重组载体导入洋葱表皮细胞,用荧光显微镜和激光共聚焦显微镜观察。【结果】与导入pCambia1300-bar载体的洋葱表皮细胞相比较,导入重组载体pCambia1300-cbcor15a-bar的洋葱表皮细胞内有强烈的绿色荧光信号。【结论】重组甘蔗转基因二元植物表达载体启动子Ubi-1能够调控下游冷诱导基因Cbcor15a和报告基因eGFP的正常高效表达,为外源基因Cbcor15a转化甘蔗提供保障。

[Objective]A new bivalent plant expression vector,named pCambia1300-cbcor15a-bar,was recombined by inserting two genes,i.e.Cbcor15a and eGFP,into the vector pCambia1300-bar and replacing the promoter CaMV 35s with Ubi-1.[Method]Based on the multiple cloning sites of the expression vector pCambia1300-bar,the primers were designed according to the nucleotide sequence of gene Cbcor15a and eGFP and the promoter Ubi-1,and then the fragments of the genes and promoter were inserted into the vector pCambia1300-bar.The plasmids of the vector pCambia1300-cbcor15a-bar were transduced into onion epidermal cells via particle bombardment.The results were observed through fluorescence microscopy and confocal laser scanning microscope,respectively.[Result]Compared to onion epidermal cell with pCambia1300-bar,onion epidermal cell transduced with the recombinant vector plasmid had a very bright green florescence.[Conclusion]Downstream cold-induced gene Cbcor15a and reporter gene eGFP regulated by up-stream promoter Ubi-1 were expressed efficiently,which could ensure the construction of Cbcor15a.